Parallel Comparison of 4-1BB or CD28 Co-stimulated CD19-Targeted CAR-T Cells for B Cell Non-Hodgkin's Lymphoma

Abstract

CD19-targeted chimeric antigen receptor-T (CAR-T) cells with CD28 or 4-1BB (28z CAR-T and BBz CAR-T) have shown great promise to treat relapsed or refractory (r/r) B cell non-Hodgkin's lymphoma (B-NHL). However, comparison of their clinical outcomes has never been reported. This study investigated their efficacy and adverse events in B-NHL therapy. Six patients with r/r B-NHL were initially enrolled and infused with 28z or BBz CAR-T cells at a dose of 0.75-5 × 10⁵/kg. These CAR-T cells showed similar antitumor efficacies, with a complete response (CR) rate of 67% within 3 months. BBz CAR-T was well tolerated. However, severe cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome occurred in the 28z CAR-T cohort, resulting in the termination of further evaluation of 28z CAR-T. Three more patients were enrolled to investigate BBz CAR-T cells in-depth at an escalated dose (1 × 10⁶/kg). All cases achieved CR within 3 months, and only grade 1/2 adverse events occurred. This study suggests that 4-1BB is more beneficial for the clinical performance of CAR-T cells than CD28 in CD19-targeted B-NHL therapy, at least under our manufacturing process.

Keywords: B cell non-Hodgkin’s lymphoma; CD19-targeted chimeric antigen receptor-T cells; co-stimulatory domain.

Graphical abstract

Figure 1

Schematic Design of the Clinical Study, CAR-T Manufacture, and CAR Molecules (A) Clinical study design. (B) CAR-T manufacturing process. (C) Composition of BBz CAR and 28z CAR.

Figure 2

Characteristics of the Manufactured BBz CAR-T and 28z CAR-T Cells (A) The time taken to manufacture BBz CAR-T and 28z CAR-T cells for each patient. (B) The expression ratio and mean fluorescence intensity (MFI) of CARs in BBz CAR-T and 28z CAR-T cells. CAR-T cells were stained with FITC-anti-CAR scFv and analyzed using flow cytometry. (C) The copy number of BBz CAR and 28z CAR in each CAR-T cell. (D) The CD4/CD8 ratio of BBz CAR-T and 28z CAR-T cells. (E) The differentiation status of BBz CAR-T and 28z CAR-T cells. CAR-T cells were stained with APC-anti-CD45RA and APC-Cy7-anti-CD62L antibodies, and analyzed using flow cytometry. CD45RA⁺CD62L⁺, CD45RA⁻CD62L⁺, and CD45RA⁻CD62L⁻ were regarded as lowly differentiated cells. *p < 0.05; the error bars represent the SD (n = 3). D1, at the dose of ∼5 × 10⁵/kg; D2, at the dose of 1 × 10⁶/kg.

Figure 3

Comparison of Cytokine Levels in Patients after Infusion of BBz CAR-T or 28z CAR-T Cells (A) Expression of IL-6, IL-10, and IFN-γ in the peripheral blood of patients after infusion of BBz CAR-T or 28z CAR-T cells for different time intervals. (B) Peak level of IL-6, IL-10, and IFN-γ in each group of patients. (C) Time intervals to reach the peak level of IL-6, IL-10, and IFN-γ in each group of patients. **p < 0.01; the error bars represent SD (n = 3). D1, at the dose of ∼5 × 10⁵/kg; D2, at the dose of 1 × 10⁶/kg.